The Golgi complex plays a central role in post-translational processing and sorting of newly synthesized proteins and lipids in all eukaryotic cells. One of its best studied functions is oligosaccharide processing, which is precisely controlled by expression and localization of glycosyltransferases and glycosidases. Glycosylation patterns change during development, and aberrant glycosylation patterns may contribute to metastasis of tumor cells. The sorting function of the Golgi complex is also critical for normal cell function, since protein and lipid mistargeting can lead to disease. Understanding the signals and mechanisms that retain Golgi resident proteins in this organelle is the first step towards understanding Golgi complex structure and function. Earlier work identified important targeting information within transmembrane domains of Golgi resident proteins. A multidisciplinary approach will be required to understand the mechanism by which transmembrane domain signals are recognized. The specific aims are to: 1) Define the signals for Golgi subcompartment localization using as models two coronavirus M proteins that are targeted to opposite faces of the Golgi complex when expressed from cDNA. Both retention mediated by transmembrane domains and retrieval using information in the cytoplasmic domains of the proteins is expected to contribute to steady- state Golgi localization. Retention and retrieval mechanisms will be investigated. 2) Identify and characterize cytosolic machinery involved in endoplasmic reticulum to Golgi transport. Efficient export from the endoplasmic reticulum is required for targeting of early Golgi residents, and transport machinery may play a role in retention by mediating concentration. Proteins interacting with the cytoplasmic tail of a model cis Golgi resident protein will be identified, and their potential functions in Golgi localization and endoplasmic reticulum to Golgi transport will be assessed. 3) Determine the role of the lipid bilayer in localization of membrane proteins to the Golgi complex. The lipid composition of Golgi subcompartments suggests that discrete differences could be involved in protein targeting to this organelle. Interactions between specific transmembrane domains will be assessed using synthetic peptides and liposomes of defined lipid compositions. In addition, Golgi localization of resident proteins will be investigated after perturbation of different classes of lipids in intact cells.